This application seeks renewed support for a hiighily productive collaboration centered on human pluripotent stem cell research. The investigators of this POI represent a highly interactive and collaborative group. Each of the projects interacts extensively with at least two of the other Projects and Cores. Project 1 (Blau) will dissect the molecular basis of stem cell quiescence using novel in vivo and in vitro models. Project 2 (Moon) will characterize context dependent changes in Wnt signaling in human pluripotent stem cells and their differentiated mesodermal and cardiomyocyte progeny (in collaboration with Project 3). In addition. Project 2 is developing a panel of signaling reporter human pluripotent stem cell lines and these lines will prove extremely valuable to Projects 1, 3 and 4 in understanding how signaling differs between distinct pluripotent states and during differentiation. Project 3 (Murry) will test novel candidate regulators of cardiomyocyte differentiation that they identified during the current funding period. Project 4 (Reh) will interact with Projects 1 and 3 on the biology of microRNAs in quiescence and maturation. Our Cores are designed to support the projects in essential aspects of their work. Our Stem Cell Core A (Ware) will work with Projects 1, 3 and 4 to examine whether metabolites can act as drivers of distinct states of pluripotency or to direct differentiation, and will work with Projects 3 and 4 to identify methods for positioning human pluripotent stem cell lines to generate cardiomyocytes or neuroretinal cells. Our Computational Biology Core B (Ruzzo) will provide bioinformatics support for each of the projects as well as for Core A, and will integrate this information with existing genetic and medical datasets. These extensive interactions between the Projects and the Cores assure that the aggregate knowledge to be gained from this Program Project vastly exceeds the sum of its parts. RELEVANCE (See instructions): The research described in this proposal will significantly enhance our understanding of stem cell biology and will advance our ability to manipulate stem cells for therapeutic purposes